Determining which amino acid residues of a protein form the binding site for a small molecule ligand, or that are present at the interface of a protein-protein complex, is difficult to identify from the crystal structure of the apo-protein. Recently, we have developed mass spectrometric methods coupled with heavy isotope labeling for mapping the location of active sites of proteins. These methods rely on measuring changes in solvent-accessibility or protein stability in the presence and absence of ligands, as determined from the rates of exchange of solvent deuterons with amide NH, a technique referred to as amide hydrogen/deuterium exchange or amide H/D-Ex. This proposal describes development of a complementary method to amide H/D-Ex, specifically hydroxyl radical-mediated isotope exchange into aliphatic carbons of amino acid side chains, a technique referred to as alkyl H/D-Ex. While amide H/D-Ex characterizes the mobility and accessibility of the polypeptide backbone, the alkyl H/D-Ex provides complementary structural information by revealing the solvent accessibility of the side chains. The proposed research builds on previous proof of-concept research to understand the underlying chemistry of the approach. The goal of this research is to demonstrate the utility of alkyl exchange to define the small molecule-protein interfaces within various small peptide/thrombin and protein/thrombin complexes; as well as, the interactions of protein kinase A with small molecule and peptide ligands. Finally, the approach will be adapted for high-throughput analysis. The specific aims of this proposal are: 1) increase the level of deuterium incorporation to improve the dynamic range of the technique, 2) use alkyl H/D-Ex to map protein/protein interactions, 3) use allcyl H/D-Ex to map ligand-protein interactions, 4) to transfer the alkyl H/D-Ex technology that was developed at Case Western Reserve University to ExSAR, and 5) develop computational methods for data analysis. PROPOSED COMMERCIAL APPLICATION: Structure based drug design is limited by the lack of low-cost and rapid methods for the analysis of ligand-protein interactions. Alkyl H/D-Ex technology represents a structure-based drug design approach that is rapid, versatile, and low cost. This technology can be adapted to high throughput allowing it to be integrated into small molecule drug discovery programs throughout the pharmaceutical industry. ExSAR Corporation will participate in the adaptations required for high throughput and validation of the technology, while the work at the institution will focus on enhancing the chemistry of the alkyl H/D-Ex. [unreadable] [unreadable]